Generating CIK NKT cells from cord blood

ABSTRACT

Provided herein are methods and customized media compositions for culturing CIK NKT cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/260,544, filed Jan. 14, 2021, which is a national stage under 35U.S.C. § 371 of PCT application No. PCT/US2019/040145, filed Jul. 1,2019, which claims priority benefit of U.S. Provisional Application No.62/696,131 filed Jul. 10, 2018, each of which is incorporated byreference herein.

BACKGROUND

Natural killer T cells (NKT cells) represent a subset of T lymphocytesthat express natural killer (NK) cell surface markers. A subset of NKTcells, termed invariant NKT cells (iNKT), express a highly restricted Tcell receptor (TCR). Although iNKT cells play an important role inlinking innate and adaptive immune responses and have been implicated invarious diseases, such as infectious diseases, allergy, asthma,autoimmunity, and tumor surveillance (Juno et al. PLoS Pathog. 2012;8(8)), their activation typically requires CD1d-restricted lipid ligandsalpha-galactosylceramide (Gal-Cer). Procedures necessary to introduceGal-Cer (e.g., in the form of a Gal-Cer/CD1d tetramer) couldsignificantly increase the cost of the treating patients using these NKTcells and in some cases limit the scope of their therapeutical utility.Thus, a need remains for a safe and cost-effective NKT cell therapy thatcan be used to treat patients having a broad range of tissue types.

BRIEF SUMMARY

Provided herein are CIK NKT cells that can be activated in the absenceof Gal-Cer. In some embodiments, greater than 50% of the cells in theCIK NKT cell population express CD56 and CD3 and less than 10% of thecells in the population express Va24. Also provided are compositions andkits comprising a plurality of CIK NKT cells from the population.Methods of producing the CIK NKT cells and using the cells to treatcancer are also provided.

This disclosure provides a population of CIK NKT cells, wherein greaterthan 50% of the cells in the population express CD56 and CD3 and lessthan 10% of the cells in the population express Va24.

Optionally, the population of CIK NKT cells can kill a target cell inthe absence of alpha-galactosylceramide(Gal-Cer). Optionally, the targetcell is a cancer cell. The cancer cell line may be selected from thegroup consisting of a myelogenous leukemia cell, a medulloblastoma cell,and a monocytic cell. Optionally, the cancer cell is selected from thegroup consisting of a K562 cell, a Daudi cell, a DAOY cell, and a THP-1cell.

Optionally, the CIK NKT cells kill a plurality of the target cells at anEC50 of between 1.0 and 10.0. Optionally, the CIK NKT cells can kill thetarget cells at a EC50 that is no less than 90% and no greater than 110%of the IC50 at which the CIK NKT cells killing the target cells in thepresence of Gal-Cer.

This disclosure also provides a composition comprising a plurality ofCIK NKT cells from any of the populations of CIK NKT cells describedabove, and a physiologically acceptable excipient.

Also provided is a kit for treating cancer comprising a plurality of CIKNKT cells from any of the populations of CIK NKT cells described aboveand a container and/or a label indicating the kit is for treatingcancer.

Also provided is a method of enriching CIK NKT cells from a cord bloodsample, the method comprising: isolating mononuclear cells from the cordblood sample; and contacting the isolated mononuclear cells with one ormore agents selected from the group consisting of IL-7, ALT-803 orIL-15, FLT3 ligand, and Gal-Cer, whereby enriching CIK NKT cells. IL-7,if present, may be in a concentration ranging from 5 to 20 ng/mL.ALT-803, if present, may be in a concentration ranging from 100 to 300ng/mL. FLT3 ligand, if present, may be in a concentration ranging from 5to 20 ng/mL. Gal-Cer, if present, may be in a concentration ranging from2 to 10 μg/mL.

Optionally, the method further comprises isolating the enriched CIK NKTcells from the rest of the cord blood sample. Optionally, the methodfurther comprises contacting the isolated CIK NKT cells with anti-CD3,anti-CD28, and IL2 to expand the CIK NKT cells. Optionally, the methodfurther comprises contacting the separated CIK NKT cells with Gal-Cer.Optionally, the Gal-Cer is used in a form of a Gal-Cer loaded CD1dtetramer. Optionally, the anti-CD3 antibody may be present in an amountof 5 ng/mL to 60 ng/mL. Optionally, the anti-CD28 antibody is present inan amount of 0.1 μg/mL to 2 μg/mL. Optionally, IL-2 is present in aconcentration of 50 ng/mL to 500 ng/mL. Optionally, enriching and/orexpansion of CIK NKT cells does not include interferon-gamma.

Also provided is a population of CIK NKT cells produced by the methodsof enriching, isolating and expanding CIK NKT cells described above.

Also provided is a method of treating cancer or viral infection in apatient in need thereof, the method comprising administering to thepatient a therapeutically effective amount of CIK NKT cells from any ofthe populations of CIK NKT cells as described above, thereby treatingcancer. Optionally, 1×10⁸ to about 1×10¹¹ CIK NKT cells per m² of bodysurface area of the patient are administered to the patient. Optionally,the cancer is selected from the group consisting of a leukemia, alymphoma, polycythemia vera, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, a sarcoma and a carcinoma.Optionally the cells are administered to the patient by a route selectedfrom the group consisting of intravenous, intraperitoneal, andsubcutaneous. Optionally, the method further comprises administering anantibody.

Also provided is a population of CIK NKT cells of claim 1, wherein theCIK NKT cells express a CAR and/or a cytokine, and greater than 50% ofthe cells in the CIK NKT cell population express CD56 and CD3 and lessthan 10% of the cells in the population express Va24.

The foregoing general description and the following detailed descriptionare exemplary and explanatory and are intended to provide furtherexplanation of the disclosure. Other objects, advantages and novelfeatures will be readily apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, features and advantages will be more readily appreciatedupon reference to the following disclosure when considered inconjunction with the accompanying drawings.

FIG. 1 is a schematic representation of the pathway which CIK NKT cellsemploy to kill target cells.

FIG. 2A shows the results of flow cytometry analysis of CIK NKT cellsstained with CD3, CD56, and Va24. FIG. 2A is the forward scatter andsize scatter diagram; FIG. 2B shows CD3 and CD56 diagrams; and FIG. 2Cshows the Va24 diagram.

FIG. 3A shows the killing of DAOY cells in the presence (represented bycircles) or absence (represented by squares) by the cord blood CIK NKTcells. FIG. 3B shows the killing of luciferase-expressing THP1 cells bycord blood CIK NKT cells (represented by squares) or peripheral bloodiNKT cells (represented by circles).

FIG. 4 shows the killing of K562 cells, DAOY cells, and Daudi cells bycord blood CIK NKT cells.

DETAILED DESCRIPTION

Overview

This application provides cytokine induced killer NKT cells (CIK NKTcells) that can kill target cells in a non-CD1d restricted manner, i.e.,independent of the formation of the Gal-Cer/CD1d tetramer. The CIK NKTcells can be used to target a broad range of target cells and will nottrigger Graft-versus-host disease (GVHD). GVHD occurs due to invasiveability of lymphocytes to infiltrate and cause extensive inflammation inorgans such as the gut, skin and liver. It has been shown that CIK NKTsdo not express chemokine receptors important for targeting to GVHDorgans but do express receptors that facilitate homing to tumors, thusthey will not trigger GVHD. As compared to existing technologies ofgenerating CIK NKT cells, which normally produce a heterogenouspopulation consisting of CD3+, CD56+ single positive cells andCD3+/CD56+ double positive cells, and the double positive cellstypically 30% or less, the present methods produce a cell populationthat consists of predominantly CD3+/CD56+ double positive cells. In oneaspect, at least 50% of the CIK NKT cells in the population express bothCD56 and CD3 and less than 10% of the cells in the population expressVa24. The method of producing CIK NKT cells do not require the exposingthe cells to interferon-gamma, which saves cost. Thus, the applicationprovides a safe and cost-effective NKT cell therapy that can be usedbroadly to treat various diseases, e.g., cancers, without causingclinically adverse symptoms such as GVHD.

The disclosure also provides compositions and kits comprising aplurality of CIK NKT cells from the population. Methods of producing theCIK NKT cells and using the CIK NKT cells to treat cancer are alsoprovided.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise. Thus, forexample, reference to “a natural killer cell” includes a plurality ofnatural killer cells.

All numerical designations, e.g., pH, temperature, time, concentration,amounts, and molecular weight, including ranges, are approximationswhich are varied (+) or (−) by increments of 0.1 or 1.0, whereappropriate. It is to be understood, although not always explicitlystated, that all numerical designations may be preceded by the term“about.”

As used herein, “+”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is detectably present influorescence activated cell sorting over an isotype control; or isdetectable above background in quantitative or semi-quantitative RT-PCR.

As used herein, “−”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is not detectablypresent in fluorescence activated cell sorting over an isotype control;or is not detectable above background in quantitative orsemi-quantitative RT-PCR.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

It is also to be understood, although not always explicitly stated, thatthe reagents described herein are merely exemplary and that equivalentsof such are known in the art.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of,” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claims. “Consisting of” shall mean excludingmore than trace amount of other ingredients and substantial methodsteps. Embodiments defined by each of these transition terms are withinthe scope of the disclosure.

As used herein, the terms “cytotoxic” and “cytolytic”, when used todescribe the activity of effector cells such as NK cells, are intendedto be synonymous. In general, cytotoxic activity relates to killing oftarget cells by any of a variety of biological, biochemical, orbiophysical mechanisms. Cytolysis refers more specifically to activityin which the effector lyses the plasma membrane of the target cell,thereby destroying its physical integrity. This results in the killingof the target cell. Without wishing to be bound by theory, it isbelieved that the cytotoxic effect of NK cells is due to cytolysis.

The term “kill” with respect to a cell/cell population is directed toinclude any type of manipulation that will lead to the death of thatcell/cell population.

The term “cytokine” or “cytokines” refers to the general class ofbiological molecules which effect cells of the immune system. Exemplarycytokines include but are not limited to FLT3 ligand, interferons andinterleukins (IL), in particular IL-2, IL-12, IL-15, IL-18 and IL-21.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “treating” or “treatment” covers the treatment of a disease ordisorder described herein, in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisorder; (iii) slowing progression of the disorder; and/or (iv)inhibiting, relieving, or slowing progression of one or more symptoms ofthe disease or disorder. The term “administering” or “administration” ofa monoclonal antibody or a natural killer cell to a subject includes anyroute of introducing or delivering the antibody or cells to perform theintended function. Administration can be carried out by any routesuitable for the delivery of the cells or monoclonal antibody. Thus,delivery routes can include intravenous, intramuscular, intraperitoneal,or subcutaneous delivery. In some embodiments the CIK NKT cells areadministered directly to the tumor, e.g., by injection into the tumor.In some embodiments the modified CIK NKT cells described herein areadministered parenterally, e.g., by injection, infusion or implantation(subcutaneous, intravenous, intramuscular, intravesicularly, orintraperitoneal).

The term “expression” refers to the production of a gene product.

As used herein, the terms “cytotoxic” when used to describe the activityof effector cells such as NK cells, relates to killing of target cellsby any of a variety of biological, biochemical, or biophysicalmechanisms.

The terms “decrease,” “reduced,” “reduction,” and “decrease” are allused herein to refer to a decrease by at least 10% as compared to areference level, for example a decrease by at least about 20%, or atleast about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% decrease (i.e. absentlevel as compared to a reference sample), or any decrease between10-100% as compared to a reference level.

The term “cancer” refers to all types of cancer, neoplasm, or malignanttumors found in mammals, including leukemia, carcinomas and sarcomas.Exemplary cancers include cancer of the brain, breast, cervix, colon,head & neck, liver, kidney, lung, non-small cell lung, melanoma,mesothelioma, ovary, sarcoma, stomach, uterus and medulloblastoma.Additional examples include, Hodgkin's Disease, Non-Hodgkin's Lymphoma,multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma,primary thrombocytosis, primary macroglobulinemia, primary brain tumors,cancer, malignant pancreatic insulanoma, malignant carcinoid, urinarybladder cancer, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, endometrial cancer, adrenal corticalcancer, neoplasms of the endocrine and exocrine pancreas, and prostatecancer.

The term “therapeutically effective amount” or “effective amount” refersto the amount required to ameliorate the symptoms of a disease relativeto an untreated patient. The effective amount of active compound(s) usedto practice the present disclosure for therapeutic treatment of adisease varies depending upon the manner of administration, the age,body weight, and general health of the subject. Ultimately, theattending physician or veterinarian will decide the appropriate amountand dosage regimen. Such amount is referred to as an “effective” amount.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent disclosure. Additionally, some terms used in this specificationare more specifically defined below.

Cytokine Induced Killer Natural Killer T Cells (CIK NKT Cells)

Natural killer T cells are a heterogeneous group of T cells that shareproperties of both T cells and natural killer cells. For example, NKTcells express an alpha beta T-cell receptor, but also express a varietyof molecular markers that are typically associated with NK cells, suchas NKp44, NKp46, and NKp30. NKT cells constitute only about 0.1% of allperipheral blood T cells. NKT cells have been implicated in suppressionof autoimmunity and graft rejection, promotion of resistance topathogens, and promotion of tumor immunity.

NKT cells are typically classified into type I and type II, based ondifferences in T cell receptor (TCR) usage. Type 1 NKT cells, alsocommonly referred to as invariant NKT cells, are NKT cells that expressa highly restricted T cell receptor, which comprises an invariant TCRalpha chain, Va24.

Invariant NKT cells are typically activated by recognizing lipid ligandsalpha-galactosylceramide (Gal-Cer) presented by CD1d. CD1d is a memberof CD1 family of glycoproteins expressed on the surface of variousantigen presenting cells and are involved in the presentation of lipidantigens. In contrast to class I and II major histocompatibility complex(MHC) molecules that present protein antigens to CD8+ and CD4+ T cells,respectively, CD1 molecules can capture and process both foreign andself lipid antigens for display to T cells. Gal-Cer are typicallyderived from pathogenic cells, for example, bacteria.

As compared to Type I NKT cells, type II NKT cells have a more diverseTCR repertoire and a higher sequence diversity. Type II NKT cells do notrespond to Gal-Cer, i.e., their activation is independent of thepresence of Gal-Cer.

The CIK NKT cells disclosed herein belong to the category of type II NKTcells. The CIK NKT cells can be produced through cytokine induction. Insome instances, they are produced from e.g., cord blood, throughcytokine induction. In some instances they are produced during theprocess of enriching or isolating iNKT cells from cord blood samples.However, CIK NKT cells differ from the typical iNKT cells in severalaspects. Phenotypically, unlike iNKT cells, which express Va24, a markerfor the alpha chain of a TCR receptor, and the percentage of iNKT cellsthat express Va24 is can be 70% or higher, CIK NKT cells have reducedexpression of Va24. For example, the percentage of CIK NKT cells thatexpress Va24 may be less than 10% of the total population of CIK NKTcells. In terms of cytotoxicity, unlike iNKT cells, which are activatedby recognizing the alpha galactosyl ceramide (Gal-Cer), a glycolipid,the CIK NKT cells do not need Gal-Cer to be activated and can kill thecells in the absence of (Gal-Cer). See FIG. 3 and FIG. 4.

Thus, in terms of phenotypes, the CIK NKT cells provided hereintypically have high expression levels of CD56 and CD3 and low expressionof Va24. In some instances, at least 90% of the CIK NKT populationproduced from the cord blood cells express CD56 and CD3, and less than10% of the cells in the population express Va24.

Method of Isolating and Culturing CIK NKT Cells

Collecting Cord Blood

Human umbilical cord blood has high composition of hematopoietic stemcells and can be used as a source for generating CIK NKT cells. Tocollect cord blood, generally, a human placenta is recovered shortlyafter its expulsion after birth. The placenta can be transported in asterile, thermally insulated transport device (maintaining thetemperature of the placenta between 20-28° C.), for example, in a cordblood collection kit substantially as described in U.S. Pat. No.7,147,626. Preferably, the placenta is delivered to the laboratory fourto twenty-four hours following delivery.

The placenta can be subjected to a conventional cord blood recoveryprocess. Typically a needle or cannula is used, with the aid of gravity,to exsanguinate the placenta (see, e.g., Anderson, U.S. Pat. No.5,372,581; Hessel et al., U.S. Pat. No. 5,415,665). The needle orcannula is usually placed in the umbilical vein and the placenta can begently massaged to aid in draining cord blood from the placenta. Suchcord blood recovery may be performed commercially, e.g., LifeBank Inc.,Cedar Knolls, N.J., ViaCord, Cord Blood Registry and CryoCell.Preferably, the placenta is gravity drained without further manipulationso as to minimize tissue disruption during cord blood recovery.

Methods for collecting cord blood cells are well known, for example, asdescribed in US20150225697. Cord blood mononuclear cells (Comics) can beisolated from collected cord blood using methods well known in the art,e.g., a density gradient method using Ficoll-Paque. Reagents suitablefor isolating Comics are commercially available, e.g., from Stem cellTechnology Inc.

Enriching CIK NKT Cells

Comics can be cultured for a period of time in the presence of variouscytokines in order to enrich for CIK NKT cells. Enriching refers toincreasing the percentage of number of target cells in a heterogenouscell population (e.g., the Comics). The enrichment period may be 2days-3 weeks, e.g., 1-2 weeks, 5-10 days, or about 2 weeks. Variousgrowth media can be used, for example, Roswell Park Memorial Institutemedium (RPMI), or Dulbecco's modified eagle medium (DMEM). Optionally,the medium further comprises human AB serum and/or Gal-Cer. Optionally,the human AB serum is present in 5-15% v/v, e.g., about 10% v/v.Optionally, the Gal-Cer is present in a concentration of 2-10 μg/mL,e.g., about 5 μg/mL. Suitable cytokines that can be added to the mediummay include one or more cytokines selected from the group consisting ofstem cell factor, FLT3 ligand, IL-7, and ALT-803 or IL-15. In someembodiments, FLT3 ligand is present in a concentration ranging from 5-20ng/mL, e.g., 10 ng/mL; IL-7 is present in a concentration ranging from5-20 ng/mL, e.g., 10 ng/mL; and /or ALT-803 is present in aconcentration ranging from 100-300 ng/mL, e.g., about 175 ng/mL.

FLT3 ligand is a hematopoietic four helical bundle cytokine and it isstructurally homologous to stem cell factor (SCF) and colony stimulatingfactor 1 (CSF-1). In synergy with other growth factors, FLT3 ligandstimulates the proliferation and differentiation of various blood cellprogenitors. It is a major growth factor stimulating the growth ofdendritic cells.

ALT-803 is a complex consisting of human IL-15 mutant IL-15N72D (residuesubstitution at position 72) and IL-15Rα sushi-Fc fusion protein (seeZhu et al. J. Immunol. 2009; 183:3598-607, the relevant disclosure ishereby incorporated by reference).

CIK NKT cells can be isolated from the enriched culture described aboveby methods well known in the art, for example, incubating magnetic beadscoupled with antibody against the Va24-J18 chain of the TCR with theenriched culture so that the CIK NKT cells will bind to the magneticbeads, and subsequently isolating the CIK NKT cells that are bound tothe beads in presence of a magnetic field. One example of the suitableantibodies that can be used is the 6B11 antibody, which are commerciallyavailable for vendors such as Biolegend (San Diego, Calif.). Suitablereagents for isolating CIK NKT cells are from Miltenyi Biotec, Germany.CIK NKT cells are typically isolated in PBS-containing serum (e.g.,human AB serum). Optionally, the isolation solution also contains EDTA.

Thus, provided herein is a method of enriching CIK NKT cells from cordblood, the method comprising: from a cord blood sample, the methodcomprising: isolating mononuclear cells from the cord blood sample; andcontacting the isolated monocytes with one or more agents selected fromthe group consisting of IL-7, ALT-803, FLT3 ligand, and Gal-Cer toenrich CIK NKT cells.

Expanding CIK NKT Cells

The CIK NKT cells as isolated above can be expanded in suitable growthmedium. Expanding refers to growing an isolated population of targetcells so that the target cells increase in number. In some embodiments,the growth medium is the NK Macs medium, available from Miltenyi Biotec,Germany. In some embodiments, the growth medium is supplemented withIL-2, anti-CD3, and/or anti CD28 antibodies in amounts suitable for NKTcell growth. In some embodiments, the anti-CD3 antibody is present in aconcentration of 5 ng/mL to 60 ng/mL, e.g., 20 ng/mL. In someembodiments, the anti-CD28 antibody is present in a concentration of 0.1μg/mL to 2 μg/mL, e.g., 0.5 μg/mL. In some embodiments, IL-2 is presentin a concentration of 50 ng/mL to 500 ng/mL, e.g., 200 ng/mL. In someembodiments, the growth medium comprises human AB serum (e.g., about 10%v/v). In some embodiments, the growth medium further comprises a Gal-Cerloaded CD1d tetramer. In some embodiments, the Gal-Cer loaded CD1dtetramer is a pre-assembled tetramer that are commercially available,e.g., from ProImmune (Oxford, UK). Methods for assembling Gal-Cer loadedCD1d tetramer is well known. Typically, Gal-Cer lipid is co-incubatedwith CD1d protein, which are oligomerized on streptavidin surface tobecome tetramers. Upon Gal-Cer binding to CD1d complex, it was columnpurified and used as reagents for expansion. Some of the exemplarymethods of preparing the Gal-Cer loaded CD1d tetramers are described inwww.proimmune.com/ecommerce/pdf_files/PS_DE000-RPE_V1.1%20%28CD1d%20Tetramer%20Empty%20%28R-PE%20Labeled%29%29.pdfand proimmune.com/ecommerce/pdf_files/ST14.pdf. In some embodiments, theGal-Cer loaded CD1d tetramer is used at an amount such that theconcentration of the Gal-Cer in the growth medium is about 20-200 ng/mL,e.g., 50-150 ng/mL, or 80-120 ng/mL, or about 100 ng/mL of Gal-Cer. Insome embodiments, the CIK NKT cells are let grown and expanded for overa few days or weeks to reach a suitable amount of cells for variousapplications.

Accordingly, the disclosure provides a method of growing CIK NKT cellsfrom cord blood, the method comprising: from a cord blood sample, themethod comprising: isolating mononuclear cells from the cord bloodsample; and contacting the isolated monocytes with one or more agentsselected from the group consisting of IL-7, ALT-803, FLT3 ligand, andGal-Cer to enrich CIK NKT cells.

Phenotyping the CIK NKT Cells

In certain embodiments, a CIK NKT cell populations can be assessed bydetecting one or more functionally relevant markers, for example, CD56and CD3 (markers for NKT cells) and TCR receptor Va24 (a high expressionof which indicates the NKT cells are invariant NKT cells).

In some embodiments, provided herein are a CIK NKT cell populationcomprising a lower percentage of Va24+ cells as compared to typicalinvariant NKT cells. The CIK NKT cell population comprises about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% ofVa24+cells. In some embodiments, the CIK NKT cell population comprisesbetween 0-20%, 5-10%, 1-7%, or 4-8% Va24+ cells. In some embodiments,the CIK NKT cell population comprises no more than 20%, no more than15%, no more than 10% of the Va24+ cells.

In some embodiments, provided herein are a CIK NKT cell populationcomprising a percentage of CD56+ cells that is substantially similar tothat in a typical invariant NKT cell population. The CIK NKT cellpopulation comprises at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or about 99%of CD56+cells. In some embodiments, the CIK NKT cell populationcomprises between 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or98-100% CD56+ cells. In some embodiments, the CIK NKT cell populationcomprises no less than 50%, no less than 70%, no less than 85%, no lessthan 90%, no less than 93%, or no less than 95% of the CD56+ cells.

In some embodiments, provided herein are a CIK NKT cell populationcomprising a percentage of CD3+ cells that is substantially similar tothat in a typical invariant NKT cell population. The CIK NKT cellpopulation comprises at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or about 99%of CD3+cells. In some embodiments, the CIK NKT cell population comprisesbetween 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or 98-100% of theCD3+ cells. In some embodiments, the CIK NKT cell population comprisesno less than 50%, no less than 70%, no less than 85%, no less than 90%,no less than 93%, or no less than 95% of the CD3+ cells.

In some embodiments, provided herein are a CIK NKT cell populationcomprising a percentage of CD56+ CD3+cells that is substantially similarto that in a typical invariant NKT cell population. The CIK NKT cellpopulation comprises at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or about 99%of CD56+CD3+cells. In some embodiments, the CIK NKT cell populationcomprises between 50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or98-100% of the CD56+CD3+cells. In some embodiments, the CIK NKT cellpopulation comprises no less than 50%, no less than 70%, no less than85%, no less than 90%, no less than 93%, or no less than 95% of theCD56+ CD3+cells.

Cytotocicity of the CIK NKT Cells

Optionally, the cytotoxic activity isolated or enriched natural killercells can be assessed, e.g., in a cytotoxicity assay using tumor cells,e.g., cultured K562, DAOY, THP-1, LN-18, U937, WERI-RB-1, U-118MG,HT-29, HCC2218, KG-1, or U266 tumor cells, or the like as target cells.CIK NKT cells disclosed herein can kill target cells regardless of MHCtype and regardless of the presence of αGal-Cer.

Assays for evaluating cytotoxicity are well known, for example, MTTassay. This is a system based on the tetrazolium compound MTT. Briefly,after the treatment period in which the target cells are in contact withCIK NKT cells, 10 uL of a freshily diluted MTT solution (2.5 mg mL⁻¹)was added to each well, and the plate was incubated at 37 C in ahumidified 5% CO2 atmosphere for 4 h. At the end of the incubationperiod, the medium was removed, and the formazan product was dissolvedin 100 μL of dimethyl sulfoxide. Cell viability was evaluated bymeasurement of the absorbance at 570 nm, using a SUNRICE Tecanabsorbance reader (Schoeller). Compound concentrations that produce 50%cell growth inhibition (IC50) were calculated from curves constructed byplotting cell survival (%) versus drug concentration (μM). The readingvalues were converted to the percentage of the control (percentage cellsurvival). Non-limiting methods of cell killing assays includeSulphorhodamine B (SRB) assay, Neutral red (NR) assay, such as thosedescribed in www.rsc.org/suppdata/mt/c4/c4mt00112e/c4mt00112e1.pdf,herein incorporated by reference in its entirety.

The efficacy of the CIK NKT cells on killing target cells can beevaluated with an EC50. EC50 used in this disclosure refers to theeffector to target ratio used in an assay where 50% of target cells arekilled. In some embodiments, the CIK NKT cells is able to kill aplurality of the target cells at an EC50 of 1-10, e.g., 1-8, 2-6, 2-5.5,or 3-7. In some embodiments, the target cell is THP-1 and the EC50 is4.64. In some embodiments, the target cell is DAOY and the IC50 is 3.69.In some embodiments, the target cell is K562 and the IC50 is 2.6.

Modified CIK NKT Cells

Chimeric Antigen Receptor

The CIK NKT cells produced as above can be further engineered to expressa chimeric antigen receptor (CAR) on the cell surface. Optionally, theCAR is specific for a tumor-specific antigen. Tumor-specific antigensare described, by way of non-limiting example, in US 2013/0189268; WO1999024566 A1; U.S. Pat. No. 7,098,008; and WO 2000020460 A1, each ofwhich is incorporated herein by reference in its entirety.Tumor-specific antigens include, without limitation, NKG2D, CS1, GD2,CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE,LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUCSAC, c-Met, EGFR, FAB,WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19 and CD33. Additionalnon-limiting tumor-associated antigens, and the malignancies associatedtherewith, can be found in Table 1.

TABLE 1 Tumor-Specific Antigens and Associated Malignancies TargetAntigen Associated Malignancy α-Folate Receptor Ovarian Cancer CAIXRenal Cell Carcinoma CD19 B-cell Malignancies Chronic lymphocyticleukemia (CLL) B-cell CLL (B-CLL) Acute lymphoblastic leukemia (ALL);ALL post Hematopoietic stem cell transplantation (HSCT) Lymphoma;Refractory Follicular Lymphoma; B-cell non-Hodgkin lymphoma (B-NHL)Leukemia B-cell Malignancies post-HSCT B-lineage Lymphoid Malignanciespost umbilical cord blood transplantation (UCBT) CD19/CD20 LymphoblasticLeukemia CD20 Lymphomas B-Cell Malignancies B-cell Lymphomas Mantle CellLymphoma Indolent B-NHL Leukemia CD22 B-cell Malignancies CD30Lymphomas; Hodgkin Lymphoma CD33 AML CD44v7/8 Cervical Carcinoma CD138Multiple Myeloma CD244 Neuroblastoma CEA Breast Cancer Colorectal CancerCS1 Multiple Myeloma EBNA3C EBV Positive T-cells EGP-2 MultipleMalignancies EGP-40 Colorectal Cancer EpCAM Breast Carcinoma Erb-B2Colorectal Cancer Breast Cancer and Others Prostate Cancer Erb-B 2, 3, 4Breast Cancer and Others FBP Ovarian Cancer Fetal Acetylcholine ReceptorRhabdomyosarcoma GD2 Neuroblastoma GD3 Melanoma GPA7 Melanoma Her2Breast Carcinoma Ovarian Cancer Tumors of Epithelial Origin Her2/newMedulloblastoma Lung Malignancy Advanced Osteosarcoma GlioblastomaIL-13R-a2 Glioma Glioblastoma Medulloblastoma KDR Tumor Neovasculaturek-light chain B-cell Malignancies B-NHL, CLL LeY Carcinomas EpithelialDerived Tumors L1 Cell Adhesion Molecule Neuroblastoma MAGE-A1 MelanomaMesothelin Various Tumors MUC1 Breast Cancer; Ovarian Cancer NKG2DLigands Various Tumors Oncofetal Antigen (h5T4) Various Tumors PSCAProstate Carcinoma PSMA Prostate/Tumor Vasculature TAA Targeted by mAbIgE Various Tumors TAG-72 Adenocarcinomas VEGF-R2 Tumor Neovasculature

In some embodiments, the CAR targets CD19, CD33 or CSPG-4.

In examples, variant polypeptides are made using methods known in theart such as oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR mutagenesis. Site direct mutagenesis (Carter,1986; Zoller and Smith, 1987), cassette mutagenesis, restrictionselection mutagenesis (Wells et al., 1985) or other known techniques canbe performed on the cloned DNA to produce CD16 variants (Ausubel, 2002;Sambrook and Russell, 2001).

Optionally, the CAR targets an antigen associated with a specific cancertype. Optionally, the cancer is selected from the group consisting ofleukemia (including acute leukemias (e.g., acute lymphocytic leukemia,acute myelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors including, but notlimited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.

In some embodiments, a polynucleotide encoding a CAR is mutated to alterthe amino acid sequence encoding for CAR without altering the functionof the CAR. For example, polynucleotide substitutions leading to aminoacid substitutions at “non-essential” amino acid residues can be made inthe CARs disclosed above. CARs can be engineered as described, forexample, in Patent Publication Nos. WO 2014039523; US 20140242701; US20140274909; US 20130280285; and WO 2014099671, each of which isincorporated herein by reference in its entirety. Optionally, the CAR isa CD19 CAR, a CD33 CAR or CSPG-4 CAR.

Additional Modifications—Cytokines

In some embodiments, CAR-expressing CIK NKT cells are further modifiedto express at least one cytokine. In specific embodiments, the at leastone cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or a variant thereof Inpreferred embodiments, the cytokine is IL-12. A representativepolypeptide of IL-12 comprises or consists of an amino acid sequence setforth in Accession No. IF45_A(https://www.ncbi.nlm.nih.gov/protein/1F45_A) and an amino acid sequenceset forth in Accession No. IF45_B(https://www.ncbi.nlm.nih.gov/protein/1F45_B).

Therapeutic Applications

This disclosure also provides a method to treat any type of cancer in asubject at any stage of the disease. Non-limiting examples of thesuitable cancers include carcinoma, melanoma, or sarcoma. In someembodiments, the invention is used to treat cancer of hemopoietic originsuch as leukemia or lymphoma. In some embodiments, the cancer is a solidtumor.

In some embodiments, the method to treat any type of cancer in a subjectcomprises administering to the patient a therapeutically effectiveamount of CIK NKT cells, wherein the thereby treating cancer. The CIKNKT cells are from a population of CIK NKT cells, wherein greater than90% of the cells in the population express CD56 and CD3 and less than10% of the cells in the population express Va24.

The disclosure also provides a method to treat any type of viralinfection, the method comprising administering to the patient atherapeutically effective amount of CIK NKT cells, wherein the therebytreating cancer. The CIK NKT cells are from a population of CIK NKTcells, wherein greater than 90% of the cells in the population expressCD56 and CD3 and less than 10% of the cells in the population expressVa24.

Also provided are methods of treating a subject in need thereof with CIKNKT cells as described herein. In some embodiments, the subject orpatient is suffering from cancer or an infectious disease, such as aviral infection.

The CIK NKT cells can be administered to an individual by absolutenumbers of cells, e.g., said individual can be administered from about1000 cells/injection to up to about 10 billion cells/injection, such asat about, at least about, or at most about, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶,5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³ (and so forth) CIK NKTcells per injection, or any ranges between any two of the numbers, endpoints inclusive. Therefore, this disclosure also provides a compositioncomprising a plurality of CIK NKT cells, wherein the number of cells are1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, or5×10³ (and so forth).

In other embodiments, said individual can be administered from about1000 cells/injection/m² to up to about 10 billion cells/injection/m²,such as at about, at least about, or at most about, 1×10⁸/m², 1×10⁷/m²,5×10⁷/m², 1×10⁶/m², 5×10⁶/m², 1×10⁵/m², 5×10⁵/m², 1×10⁴/m², 5×10⁴/m²,1×10³/m², 5×10³/m² (and so forth) CIK NKT cells per injection, or anyranges between any two of the numbers, end points inclusive.

In other embodiments, CIK NKT cells can be administered to suchindividual by relative numbers of cells, e.g., said individual can beadministered about 1000 cells to up to about 10 billion cells perkilogram of the individual, such as at about, at least about, or at mostabout, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴,1×10³, or 5×10³ (and so forth) CIK NKT cells per kilogram of theindividual, or any ranges between any two of the numbers, end pointsinclusive.

In other embodiments, the total dose may be calculated by m² of bodysurface area, including about 1×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, perm², or any ranges between any two of the numbers, end points inclusive.The average person is about 1.6 to about 1.8 m². In a preferredembodiment, between about 1 billion and about 3 billion CIK NKT cellsare administered to a patient. In other embodiments, the amount of CIKNKT cells injected per dose may calculated by m² of body surface area,including 1×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, per m². The average bodysurface area for a person is 1.6-1.8 m².

In other embodiments, CIK NKT cells can be administered to suchindividual by relative numbers of cells, e.g., said individual can beadministered about 1000 cells to up to about 10 billion cells perkilogram of the individual, such as at about, at least about, or at mostabout, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴,1×10³, or 5×10³ (and so forth) CIK NKT cells per kilogram of theindividual, or any ranges between any two of the numbers, end pointsinclusive.

CIK NKT cells can be administered once to a patient with cancer or theycan be administered multiple times, e.g., once every 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours,or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more weeks during therapy, or any ranges between anytwo of the numbers, end points inclusive.

In some embodiments, CIK NKT cells are administered in a compositioncomprising the CIK NKT cells and a medium, such as human serum or anequivalent thereof. In some embodiments, the medium comprises humanserum albumin. In some embodiments, the medium comprises human plasma.In some embodiments, the medium comprises about 1% to about 15% humanserum or human serum equivalent. In some embodiments, the mediumcomprises about 1% to about 10% human serum or human serum equivalent.In some embodiments, the medium comprises about 1% to about 5% humanserum or human serum equivalent. In a preferred embodiment, the mediumcomprises about 2.5% human serum or human serum equivalent. In someembodiments, the serum is human AB serum. In some embodiments, a serumsubstitute that is acceptable for use in human therapeutics is usedinstead of human serum. Such serum substitutes may be known in the art,or developed in the future. Although concentrations of human serum over15% can be used, it is contemplated that concentrations greater thanabout 5% will be cost-prohibitive. In some embodiments, CIK NKT cellsare administered in a composition comprising CIK NKT cells and anisotonic liquid solution that supports cell viability. In someembodiments, CIK NKT cells are administered in a composition that hasbeen reconstituted from a cryopreserved sample.

Pharmaceutically aceptable compositions comprising the CIK NKT cells caninclude a variety of carriers and excipients. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Suitablecarriers and excipients and their formulations are described inRemington: The Science and Practice of Pharmacy, 21st Edition, David B.Troy, ed., Lippicott Williams & Wilkins (2005). By pharmaceuticallyacceptable carrier is meant a material that is not biologically orotherwise undesirable, i.e., the material is administered to a subjectwithout causing undesirable biological effects or interacting in adeleterious manner with the other components of the pharmaceuticalcomposition in which it is contained. If administered to a subject, thecarrier is optionally selected to minimize degradation of the activeingredient and to minimize adverse side effects in the subject. As usedherein, the term pharmaceutically acceptable is used synonymously withphysiologically acceptable and pharmacologically acceptable. Apharmaceutical composition will generally comprise agents for bufferingand preservation in storage and can include buffers and carriers forappropriate delivery, depending on the route of administration.

These compositions for use in in vivo or in vitro may be sterilized bysterilization techniques employed for cells. The compositions maycontain acceptable auxiliary substances as required to approximatephysiological conditions such as pH adjusting and buffering agents,toxicity adjusting agents and the like, for example, sodium acetate,sodium chloride, potassium chloride, calcium chloride, sodium lactateand the like. The concentration of cells in these formulations and/orother agents can vary and will be selected primarily based on fluidvolumes, viscosities, body weight and the like in accordance with theparticular mode of administration selected and the subject's needs.

In one embodiment, CIK NKT cells are administered to the patient inconjunction with one or more other treatments or agent for the cancerbeing treated. In some embodiments, the one or more other treatments forthe cancer being treated include, for example, an antibody, radiation,chemotherapeutic, stem cell transplantation, or hormone therapy.

In some embodiments, CIK NKT cells and the other cancer agent/treatmentare administered simultaneously or approximately simultaneously (e.g.,within about 1, 5, 10, 15, 20, or 30 minutes of each other). In someembodiments, the CIK NKT cells and the other cancer agent/treatment areadministered sequentially. In some embodiments, the other cancertreatment/agent is administered one, two, or three days after theadministration of the CIK NKT cells.

In one embodiment, the other cancer agent is an antibody. In oneembodiment, CIK NKT cells are administered in conjunction with anantibody targeting the diseased cells. In one embodiment, CIK NKT cellsand an antibody are administered to the patient together, e.g., in thesame formulation; separately, e.g., in separate formulations,concurrently; or can be administered separately, e.g., on differentdosing schedules or at different times of the day. When administeredseparately, the antibody can be administered via any suitable route,such as intravenous or intra-tumoral injection.

In some embodiments, CIK NKT cells of the present disclosure are used incombination with therapeutic antibodies and/or other anti-cancer agents.Therapeutic antibodies may be used to target cells that expresscancer-associated or tumor-associated markers. Examples of cancertherapeutic monoclonal antibodies are shown in Table 2.

TABLE 2 Illustrative therapeutic monoclonal antibodies Examples ofFDA-approved therapeutic monoclonal antibodies Brand Indication Antibodyname Company Target (Targeted disease) Alemtuzumab Campath ® GenzymeCD52 Chronic lymphocytic leukemia Brentuximab Adcetris ® CD30 Anaplasticlarge cell vedotin lymphoma (ALCL) and Hodgkin lymphoma CetuximabErbitux ® Bristol-Myers epidermal growth Colorectal cancer, Head andSquibb/Eli factor receptor neck cancer Lilly/Merck KGaA GemtuzumabMylotarg ® Wyeth CD33 Acute myelogenous leukemia (with calicheamicin)Ibritumomab Zevalin ® Spectrum CD20 Non-Hodgkin tiuxetanPharmaceuticals, lymphoma (with yttrium- Inc. 90 or indium-111)Ipilimumab (MDX-101) Yervoy ® blocks CTLA-4 Melanoma OfatumumabArzerra ® CD20 Chronic lymphocytic leukemia Palivizumab Synagis ®MedImmune an epitope of the Respiratory Syncytial Virus RSV F proteinPanitumumab Vectibix ® Amgen epidermal growth Colorectal cancer factorreceptor Rituximab Rituxan ®, Biogen CD20 Non-Hodgkin lymphomaMabthera ® Idec/Genentech Tositumomab Bexxar ® GlaxoSmithKline CD20Non-Hodgkin lymphoma Trastuzumab Herceptin ® Genentech ErbB2 Breastcancer Blinatunomab bispecific CD19- Philadelphia chromosome- directedCD3 T- negative relapsed or cell engager refractory B cell precursoracute lymphoblastic leukemia (ALL) Avelumamab anti-PD-L1 Non-small celllung cancer, metastatic Merkel cell carcinoma; gastic cancer, breastcancer, ovarian cancer, bladder cancer, melanoma, meothelioma, includingmetastatic or locally advanced solid tumors Daratumumab CD38 Multiplemyeloma Elotuzumab a SLAMF7-directed (also Multiple myeloma known as CD319) immunostimulatory antibody

Administration of such CIK NKT cells may be carried out simultaneouslywith the administration of the monoclonal antibody, or in a sequentialmanner. In some embodiments, the CIK NKT cells are administered to thesubject after the subject has been treated with the monoclonal antibody.Alternatively, the CIK NKT cells may be administered at the same time,e.g., within 24 hours, of the monoclonal antibody.

In some embodiments, CIK NKT cells are administered intravenously. Insome embodiments the CIK NKT cells are infused directly into the bonemarrow.

Therefore, this disclosure provides a method of treating cancer or viralinfection in a patient in need thereof, the method comprisingadministering to the patient a therapeutically effective amount of CIKNKT cells from the population of CIK NKT cells using the methodsdisclosed herein to thereby treating cancer

Kits

Also disclosed are kits for the treatment of cancer or an infectiousdisease using compositions comprising an amount of CIK NKT cells asdescribed herein. In some embodiments, the kits of the presentdisclosure may also include at least one monoclonal antibody.

In certain embodiments, the kit may contain additional compounds such astherapeutically active compounds or drugs that are to be administeredbefore, at the same time or after administration of CIK NKT cells.Examples of such compounds include an antibody, vitamins, minerals,fludrocortisone, ibuprofen, lidocaine, quinidine, chemotherapeutic, etc.

In various embodiments, instructions for use of the kits will includedirections to use the kit components in the treatment of a cancer or aninfectious disease. The instructions may further contain informationregarding how to CIK NKT cells (e.g., thawing and/or culturing). Theinstructions may further include guidance regarding the dosage andfrequency of administration.

In certain embodiments, the kit further comprises one or more containersfilled with one or more compositions described herein, e.g., acomposition comprising CIK NKT cells as described herein. Optionallyassociated with such containers can be a label indicating the kit is fortreating a cancer, such as those described herein. Optionally the labelalso includes a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The present disclosure and the working examples exemplifies producingand using CIK NKT cells derived from cord blood samples, one or ordinaryskill in the art would appreciate that CIK NKT cells can also begenerated from other hematopoietic progenitor cell samples using similarapproaches to the ones described herein.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

EXAMPLES

This disclosure comprises the following, non-limiting embodiments.

Embodiment 1. A population of CIK NKT cells, wherein greater than 50% ofthe cells in the population express CD56 and CD3 and less than 10% ofthe cells in the population express Va24.

Embodiment 2. The population of CIK NKT cells of embodiment 1, whereinthe CIK NKT cells can kill a target cell in the absence ofalpha-galactosylceramide(Gal-Cer).

Embodiment 3. The population of CIK NKT cells of embodiment 1, whereinthe target cell is a cancer cell.

Embodiment 4. The population of CIK NKT cells of embodiment 1, whereinthe cancer cell line is selected from the group consisting of amyelogenous leukemia cell, a medulloblastoma cell, and a monocytic cell.

Embodiment 5. The population of CIK NKT cells of embodiment 3, whereinthe cancer cell is selected from the group consisting of a K562 cell, aDaudi cell, a DAOY cell, and a THP-1 cell.

Embodiment 6. The population of CIK NKT cells of embodiments 2-5,wherein the CIK NKT cells kill a plurality of the target cells at anEC50 of between 1.0 and 10.0.

Embodiment 7. The population of CIK NKT cells of embodiments 6, whereinthe CIK NKT cells can kill the target cells at a EC50 that is no lessthan 90% and no greater than 110% of the EC50 at which the CIK NKT cellskilling the target cells in the presence of Gal-Cer.

Embodiment 8. A composition comprising a plurality of CIK NKT cells fromthe population of CIK NKT cells of any of embodiments 1-7, and aphysiologically acceptable excipient.

Embodiment 9. A kit for treating cancer comprising a plurality of CIKNKT cells from the population of CIK NKT cells of any of embodiments1-7, wherein the kit further comprises a container and/or a labelindicating the kit is for treating cancer.

Embodiment 10. A method of enriching CIK NKT cells from a cord bloodsample, the method comprising: isolating mononuclear cells from the cordblood sample; and contacting the isolated mononuclear cells with one ormore agents selected from the group consisting of IL-7, ALT-803 orIL-15, FLT3 ligand, and Gal-Cer, whereby enriching CIK NKT cells.

Embodiment 11. The method of embodiment 10, wherein the IL-7, ifpresent, is in a concentration ranging from 5 to 20 ng/mL.

Embodiment 12. The method of embodiment 10 or 11, wherein the ALT-803,if present, is in a concentration ranging from 100 to 300 ng/mL.

Embodiment 13. The method of any of embodiments 10-12, wherein the FLT3ligand, if present, is in a concentration ranging from 5 to 20 ng/mL.

Embodiment 14. The method of any of embodiments 10-13, wherein theGal-Cer is present in a concentration ranging from 2 to 10 μg/mL.

Embodiment 15. The method of embodiment 10, wherein the method furthercomprises isolating the enriched CIK NKT cells from the rest of the cordblood sample.

Embodiment 16. The method of embodiment 15, wherein the method furthercomprises contacting the isolated CIK NKT cells with anti-CD3,anti-CD28, and IL2 to expand the CIK NKT cells.

Embodiment 17. The method of embodiment 16, wherein the method furthercomprises contacting the isolated CIK NKT cells with Gal-Cer.

Embodiment 18. The method of embodiment 17, wherein the Gal-Cer is apresent in a form of a Gal-Cer loaded CD1d tetramer.

Embodiment 19. The method of embodiment 16, wherein the anti-CD3antibody is present in an amount of 5 ng/mL to 60 ng/mL.

Embodiment 20. The method of any of embodiments 16-19, wherein theanti-CD28 antibody is present in an amount of 0.1 μg/mL to 2 μg/mL.

Embodiment 21. The method of any of embodiments 16-20, wherein IL-2 ispresent in a concentration of 50 ng/mL to 500 ng/mL.

Embodiment 22. The method of any of embodiments 16-21, wherein theproduction of CIK NKT cells does not include interferon-gamma.

Embodiment 23. A method of treating cancer or viral infection in apatient in need thereof, the method comprising administering to thepatient a therapeutically effective amount of CIK NKT cells from thepopulation of CIK NKT cells of any of embodiments 1-7, thereby treatingcancer.

Embodiment 24. The method of embodiment 23, wherein about 1×10⁸ to about1×10¹¹ cells per m² of body surface area of the patient are administeredto the patient.

Embodiment 25. The method of embodiment 23, wherein the cancer isselected from the group consisting of a leukemia, a lymphoma,polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, a sarcoma and a carcinoma.

Embodiment 26. The method of embodiment 25, wherein the cells areadministered to the patient by a route selected from the groupconsisting of intravenous, intraperitoneal, and subcutaneous.

Embodiment 27. The method of any of embodiments 23-26, wherein themethod further comprises administering an antibody.

Embodiment 28. A population of CIK NKT cells produced by the methods ofany of embodiments 10-27.

Embodiment 29. The population of CIK NKT cells of embodiment 1, whereinthe CIK NKT cells express a CAR and/or a cytokine.

EXAMPLES

The following examples are for illustrative purposes only and should notbe interpreted as limitations. There are a variety of alternativetechniques and procedures available to those of skill in the art whichwould similarly permit one to successfully perform the examples below.

Example 1 Phenotypes of CIK-NKT Cells

Cord blood mononuclear cells (Comics) were isolated from cord bloodsamples by density gradient method using Ficoll-Paque and Sepmate™, fromStem cell Technology Inc. Comics were incubated with Gal-Cer (5 μg/mL),FLT3-L (10 ng/mL), IL-7 (10 ng/mL), and ALT-803 (175 ng/mL) forenrichment of NKT for 2 weeks in RPMI medium with 10% Human AB serum.NKT cells were isolated by affinity chromatography using reagents fromMiltenyi Biotec, Germany. Subsequently, isolated cells were expanded inthe presence of anti-CD3 antibody (20 ng/mL), anti-CD28 antibody (0.5μg/mL), and IL2 (200 ng/mL) in the NK Macs medium with 10% Human ABserum for overnight activation with Gal-Cer loaded CD1d tetramer. TheGal-Cer loaded CD1d tetramer was from ProImmune, Oxford, UK and was usedin an amount such that the Gal-Cer was present in an amount of 100 ng/mLUpon a week of expansion, these cells were stained with antibodiesrecognizing CD3, CD56, or Va24. The results show that a majority of thecells (97.1%) were positive for both CD56 and CD3 (FIG. 2B) and a smallpercentage of cells (6.45%) were positive for Va24 (FIG. 2C). Thisindicates that cord blood CIK NKT cells have low Va24 expression but theexpression of CD3 and CD56 remain intact.

Example 2 Cytotoxicity of CIK NKT Cells is Independent of Gal-Cer

The NKT-CIK cells prepared as described in Example 2 were assessed forcytotoxicity against cancer cell line DAOY. DAOY cells were incubatedwith Gal-Cer at 1 μg/mL overnight. The Gal-Cer treated DAOY Cells werethen incubated with a fluorescent dye, Calcien AM, for 30 min. The cellswere washed and then incubated with CB-NKT CIKs, at various effector totarget ratios as indicated (the highest ratio was 32:1), for 4 hours.The killing of the DAOY cells was measured by the cell lysis, which isrepresented by the amount of CalcienAM dye released from cells. FIG. 3Ashows the amount of CalcienAM dye released from DAOY cells caused by theCB-NKT CIKs. Data were presented as % lysis of target cells. The resultsshow that there were significant difference in terms of killing betweenthe group in which target cell were loaded with Gal-Cer and the group inwhich target cells were not loaded with Gal-Cer, indicating that Gal-Certreatment did not confer killing specificity.

FIG. 3B compares the cytotoxicity of NKT-CIK cells derived from cordblood as described above (CB-CIK NKT cells) versus PBiNKT cells (iNKTcells isolated from peripheral blood) on a luciferase-expressing THP1cells. PBiNKT cells were isolated in the same manner as the CB-CIK NKTcells (see Example 1) except that the source is peripheral blood insteadof cord blood. The THP1 cells were co-cultured with CB-CIK NKT cells orPBiNKT cells at various effector to target ratios as indicated, thehighest ratio being 32:1. No Gal-Cer was used in this experiment. Theresults show that CB-CIK NKT cells are more potent in killing THP-1 thando PBiNKT cells.

Example 3

Cytotoxicity of CIK NKT Cells on K562 and Daudi Cells

CB-NKT CIK cells obtained as above were assayed for their ability tokill luciferase-expressing cancer cell lines, DAOY, Daudi, and K562(represented by triangles, squares, and circles, respectively, in FIG.4). Peripheral blood isolated iNKTs were used as control (invertedtriangles in FIG. 4). Cell killing assays were performed after 4-hour ofco-culturing of the cancer cells and effector cells (CB CIK NKTs orPBiNKTs). The killing of the cancer cells was measured by % of celllysis, which was represented by the loss of luciferase in these cancercell lines. Various effector to target ratios were used as indicated,the highest being 32:1. The results indicate that CB-CIK NKT cells killtarget cells in a non CD1d/MHC restricted manner.

What is claimed is:
 1. A method of obtaining CIK NKT cells from a cordblood sample, the method comprising: isolating mononuclear cells fromthe cord blood sample; and contacting the isolated mononuclear cellswith i) IL-7, ii) ALT-803 or IL-15, iii) FLT3 ligand, and iv) Gal-Cer,thereby producing a population of CIK NKT cells greater than 90% ofwhich express CD56 and CD3 and less than 10% of which express Va24. 2.The method of claim 1, wherein the IL-7 is present in a concentrationranging from 5 to 20 ng/mL.
 3. The method of claim 1, wherein theALT-803, if present, is in a concentration ranging from 100 to 300ng/mL.
 4. The method of claim 1, wherein the FLT3 ligand is present in aconcentration ranging from 5 to 20 ng/mL.
 5. The method of claim 1,wherein the Gal-Cer is present in a concentration ranging from 2 to 10μg/mL.
 6. The method of claim 1, wherein the method further comprisesisolating the enriched CIK NKT cells from the rest of the cord bloodsample.
 7. The method of claim 6, wherein the method further comprisesexpanding the isolated CIK NKT cells with anti-CD3, anti-CD28, and IL2.8. The method of claim 7, wherein the Gal-Cer is a present in a form ofa Gal-Cer loaded CD1d tetramer.
 9. The method of claim 7, wherein theanti-CD3 antibody is present in an amount of 5 ng/mL to 60 ng/mL. 10.The method of claim 7, wherein the anti-CD28 antibody is present in anamount of 0.1 μg/mL to 2 μg/mL.
 11. The method of claim 7, wherein IL-2is present in a concentration of 50 ng/mL to 500 ng/mL.
 12. The methodof claim 7, wherein the production of CIK NKT cells does not includeinterferon-gamma.
 13. The population of CIK NKT cells produced by themethod of claim 1, wherein the CIK NKT cells express a CAR and/or acytokine.
 14. The method of claim 7, wherein the population of expandedCIK NKT cells comprise cytotoxic effector cells that are cytotoxic inthe absence of Gal-Cer.
 15. The method of claim 7, wherein greater than90% of the population of expanded CIK NKT cells express CD56 and CD3,and less than 10% of the population express Va24.
 16. A population ofCIK NKT cells produced by the method of claim 1, wherein greater than90% of the cells in the population express CD56 and CD3 and less than10% of the cells in the population express Va24.
 17. The population ofCIK NKT cells of claim 16, wherein the CIK NKT cells can kill a targetcell in the absence of alpha-galactosylceramide(Gal-Cer).
 18. Thepopulation of CIK NKT cells of claim 17, wherein the CIK NKT cells killa plurality of the target cells at an EC50 of between 1.0 and 10.0. 19.The population of CIK NKT cells of claim 18, wherein the CIK NKT cellscan kill the target cells at a EC50 that is no less than 90% and nogreater than 110% of the EC50 at which the CIK NKT cells killing thetarget cells in the presence of Gal-Cer.
 20. The population of CIK NKTcells of claim 16, wherein the target cell is a cancer cell.
 21. Thepopulation of CIK NKT cells of claim 20, wherein the cancer cell isselected from the group consisting of a K562 cell, a Daudi cell, a DAOYcell, and a THP-1 cell.
 22. The population of CIK NKT cells of claim 16,wherein the cancer cell line is selected from the group consisting of amyelogenous leukemia cell, a medulloblastoma cell, and a monocytic cell.23. A composition comprising a plurality of CIK NKT cells from thepopulation of CIK NKT cells of claim 16, and a physiologicallyacceptable excipient.
 24. A kit for treating cancer comprising aplurality of CIK NKT cells from the population of CIK NKT cells of claim16, wherein the kit further includes a container and/or a labelindicating the kit is for treating cancer.
 25. A method of enriching CIKNKT cells from a cord blood sample, the method comprising: 1) isolatingmononuclear cells from the cord blood sample; and contacting theisolated mononuclear cells with IL-7, ALT-803 or IL-15, or FLT3 ligand,and Gal-Cer, thereby enriching the CIK NKT cells; 2) isolating theenriched CIK NKT cells from the rest of the cord blood sample; and 3)expanding the isolated CIK NKT cells with anti-CD3, anti-CD28, IL2, andGal-Cer; thereby producing a population of CIK NKT cells greater than90% of which express CD56 and CD3 and less than 10% of which expressVa24.